Several sulfur-containing amino acids (SAA) have been shown to be potent agonists of excitatory amino acid receptors in the mammalian central nervous system [Griffiths, R. (1993), Biochem. Soc. Trans. 21:66-72; Mewett et al. (1983), in Mandell, P., DeFeudis, F. V. (eds) CNS Receptors: From Pharmacology to Behaviour, 163-174, Raven Press, New York; Cox et al. (1977) J. Neurochem 29:579-588]. Homocysteine sulfinic acid (HC-SO.sub.2) and homocysteic acid (HC-SO.sub.3) are sulfur-containing homologues of glutamic acid, while cysteine sulfinic acid (C-SO.sub.2) and cysteic acid (C-SO.sub.3) are homologues of aspartic acid. These compounds are released on depolarization, bind to specific receptor sites in the brain and have been demonstrated immunocytochemically in various parts of the mammalian brain [Cox et al. (1977) supra; Luini et al. (1984) Brain Res. 324:271-277; Cuenod et al. (1990) J. Histochem. Cytochem. 38:1713-1715]. Homocysteic acid has been shown to be agonistic to pre- and post-synaptic N-methyl-D-Aspartate (NMDA) receptors [Do et al. (1986) J. Neurochem. 46:779-786; Smirnova et al. (1993) Science 262:430-433]. HC-SO.sub.2 and HC-SO.sub.3 are thought to be generated endogenously from the oxidation of homocysteine [Do et al. (1988) J.Neural. Transm. 72:185-190] while C-SO.sub.2 and C-SO.sub.3 are thought to arise endogenously from oxidation of cysteine. The structures of the sulfhydryl amino acids and the oxidized derivatives are shown in Scheme I herein.
Homocysteine itself arises predominantly from methionine through the transmethylation pathway involving S-adenosylmethionine (SAM), the universal physiologic methyl donor. Enzymatic reactions involving conversion of homocysteine to cystathionine, cystathionine to cysteine, cysteine to C-SO.sub.2 and C-SO.sub.2 to C-SO.sub.3 have been described [Mudd et al. (1989) in Scriver, C. R. et al. (eds) Disorders of Transsulfuration, p.695, New York, McGraw Hill; Ohmori et al. (1972) Physiol. Chem. & Physics. 4:286-294]. As these SAA are generally derived from endogenous homocysteine, measurement of these compounds is of interest in clinical situations with potential homocysteinemia. Homocysteinemia is known to occur in deficiency of folate, cobalamin or pyridoxine or in inborn errors of metabolism such as cystathionine .beta.-synthase deficiency or 5,10,methylenetetrahydrofolate reductase deficiency [Mudd et al. (1989) supra; Olszewski et al. (1993) Free Radical Biol. Med. 14:683-693; Stabler et al. (1988) J. Clin. Invest. 81:466-474]. Classic homocystinuria (secondary to cystathionine .beta.-synthase deficiency) leads to a variety of organ system disturbances including neuropsychiatric illness [Mudd et al. (1989) supra]. Deficiency of folate, cobalamin and pyridoxine also causes neuropsychiatric disturbances [Stabler et al. (1988) supra; Shorvon et al. (1980) Br. Med. J. 281:1036-1038; Driskell, J. A. (1984) in Machlin L. J. (Ed.) Handbook of Vitamins, p. 379, New York, Marcel Dekker]. In a study of neurosurgical biopsy samples, the concentration of homocysteic acid in brain tissue in a subgroup of patients with intractable depression was elevated as compared to other patients [Francis et al. (1989) Brain Res. 494:315-324].
HC-SO.sub.2 and HC-SO.sub.3 have been isolated from the urine of patients with cystathionine .beta.-synthase deficiency using paper chromatography [Ohmori et al. (1972) supra]. High performance liquid chromatography (HPLC) based methods utilizing a combination of external standards and retention times have been described for measurement of these compounds in brain tissue [Grieve and Griffiths (1992) Neuroscience Letters 145:1-5]. It has been suggested that a combination of internal standards and mass spectrometry is necessary for definitive identification and measurement of these SAA in tissues [Grieve and Griffiths (1992) supra; Klancnik et al. (1992) Neuroscience 49:557-570]. Measurement of these oxidation products of cysteine and homocysteine with certainty in human or animal serum is not believed to have been reported previously.
While various HPLC methods have been reported for determining levels of sulfur-containing amino acids in tissue samples, the identification of those compounds has not been absolute according to Grieve and Griffiths (1992) supra, who report that the identification was based on synchronization of retention times, "spiking" of samples with authentic markers, or a combination of those strategies. Grieve and Griffiths (1992) supra, further characterized Klancnik et al. (1992) supra, as reporting that the unequivocal identification of sulfur-containing amino acids is "currently unattainable due to a number of technical difficulties."
Thus, there is a need in the art for a sensitive and definitive assay for the measurement of homocysteine sulfinic acid, homocysteic acid, cysteine sulfinic acid and cysteic acid in biological and other samples, especially with application to the diagnosis of certain neuropsychiatric disorders of biochemical origin.